Sequence-specific RNA Recognition Research Articles

Major-groove sequence-specific RNA recognition by LoaP, a paralog of transcription elongation factor NusG

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding β-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.

Krisp: A Python package to aid in the design of CRISPR and amplification-based diagnostic assays from whole genome sequencing data.

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.

An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G

An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5’-AA-3’ dinucleotides, and to a lesser extent, 5’-GA-3’ dinucleotides. APOBEC3G binds to the common 3’A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5’A or 5’G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly

In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and invivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.

The human pseudouridine synthase PUS7 recognizes RNA with an extended multi-domain binding surface.

The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure–function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function.

Recognition of Target Site in Various Forms of DNA and RNA by Peptide Nucleic Acid (PNA): From Fundamentals to Practical Applications

Abstract Peptide nucleic acid (PNA) is a DNA analog, in which the sugar-phosphate backbone in DNA is replaced by poly[N-(2-aminoethyl)glycine]. Since its discovery in the early 1990s, PNA has been widely employed in chemistry, biochemistry, medicine, nanotechnology, and many other fields. This account surveys recent developments on the design of PNA derivatives and their applications. In the first part, PNAs for sequence-specific recognition of DNA and RNA (single-strands, double-strands, G-quadruplexes, i-motifs, and others) are comprehensively covered. Modifications of nucleobases and of the main chain effectively promote both the strength of binding and the selectivity of recognition. In the second half of this account, practical applications of PNA are presented. Structural restraints, induced by complex formation of PNA with DNA and RNA substrates, lead to selective transformation of target sites to desired structures. Applications to regulation of gene expression, gene editing, construction of sophisticated nanostructures, and others are also described. Advantages and disadvantages of PNAs, compared with other sequence-recognizing molecules hitherto reported, are discussed in terms of various physicochemical and biological features.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5′ single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3′ single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA–Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

Identification of new pentatricopeptide repeat proteins, MREF1 and 2, involved in mitochondrial RNA editing, using computational target RNA prediction.

C-to-U RNA editing has been widely observed in mitochondrial and plastid RNAs in plants. The editing sites are known to be recognized by pentatricopeptide repeat (PPR) proteins, which belong to one of the largest protein families in vascular plants. PPR proteins are sequence-specific RNA-binding proteins that participate in various steps of organelle RNA metabolism, such as cleavage, stabilization, splicing, translation, and editing. Elucidating the underlying mechanisms of sequence-specific RNA recognition by PPR proteins expanded our understanding of the role of PPR proteins in plant organellar RNA editing and enabled the computational prediction of target RNA-editing sites for PPR proteins of interest. Combining computational prediction and experimental verification, we identified three new PPR proteins involved in mitochondrial RNA editing: At1g56570, known as PGN for RNA editing of nad6_leader_-73 and cox2_742, At4g04370 for RNA editing of nad5_242, and At2g41080 for atp1_1292. Therefore, At4g04370 and At2g41080 were designated as mitochondrial RNA-editing factor 1 (MREF1) and MREF2, respectively. This study supports the use of computational prediction in establishing connections between PPR proteins and specific RNA-editing sites, which are important for maintaining various physiological processes, such as plant development, embryogenesis, and biotic- and abiotic-stress responses.

Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA.

Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

Understanding the binding specificities of mRNA targets by the mammalian Quaking protein.

Mammalian Quaking (QKI) protein, a member of STAR family of proteins is a mRNA-binding protein, which post-transcriptionally modulates the target RNA. QKI protein possesses a maxi-KH domain composed of single heterogeneous nuclear ribonucleoprotein K homology (KH) domain and C-terminal QUA2 domain, that binds a sequence-specific QKI RNA recognition element (QRE), CUAAC. To understand the binding specificities for different mRNA sequences of the KH-QUA2 domain of QKI protein, we introduced point mutations at different positions in the QRE resulting in twelve different mRNA sequences with single nucleotide change. We carried out long unbiased molecular dynamics simulations using two different sets of recently updated forcefield parameters: AMBERff14SB+RNAχOL3 and CHARMM36 (with CMAP correction). We analyzed the changes in intermolecular dynamics as a result of mutation. Our results show that AMBER forcefields performed better to model the interactions between mRNA and protein. We also calculated the binding affinities of different mRNA sequences and found that the relative order correlates to the reported experimental studies. Our study shows that the favorable binding with the formation of stable complex will occur when there is an increase of the total intermolecular contacts between mRNA and protein, but without the loss of native contacts within the KH-QUA domain.

The E domain of CRR2 participates in sequence-specific recognition of RNA in plastids.

Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. We identified a surprisingly large number of small RNAs that represent invivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both invitro and invivo experiments to reveal the role of the E domain of CRR2. The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR-RNA interaction. The 'code' of the E domain PPR motifs is different from that of P- and S-motifs. The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs invivo.

Molecular recognition of pre-tRNA by Arabidopsis protein-only Ribonuclease P.

Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5′ end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP–pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4–5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5′ leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP–substrate recognition and provides a new predictive model for the PRORP–substrate complex.

Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6

Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.

Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides.

Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available.

Deciphering the protein-RNA recognition code: combining large-scale quantitative methods with structural biology.

RNA binding proteins (RBPs) are key factors for the regulation of gene expression by binding to cis elements, i.e. short sequence motifs in RNAs. Recent studies demonstrate that cooperative binding of multiple RBPs is important for the sequence-specific recognition of RNA and thereby enables the regulation of diverse biological activities by a limited set of RBPs. Cross-linking immuno-precipitation (CLIP) and other recently developed high-throughput methods provide comprehensive, genome-wide maps of protein-RNA interactions in the cell. Structural biology gives detailed insights into molecular mechanisms and principles of RNA recognition by RBPs, but has so far focused on single RNA binding proteins and often on single RNA binding domains. The combination of high-throughput methods and detailed structural biology studies is expected to greatly advance our understanding of the code for protein-RNA recognition in gene regulation, as we review in this article.

Can We Execute Stable Microsecond-Scale Atomistic Simulations of Protein-RNA Complexes?

We report over 30 μs of unrestrained molecular dynamics simulations of six protein-RNA complexes in explicit solvent. We utilize the AMBER ff99bsc0χ(OL3) RNA force field combined with the ff99SB protein force field and its more recent ff12SB version with reparametrized side-chain dihedrals. The simulations show variable behavior, ranging from systems that are essentially stable to systems with progressive deviations from the experimental structure, which we could not stabilize anywhere close to the starting experimental structure. For some systems, microsecond-scale simulations are necessary to achieve stabilization after initial sizable structural perturbations. The results show that simulations of protein-RNA complexes are challenging and every system should be treated individually. The simulations are affected by numerous factors, including properties of the starting structures (the initially high force field potential energy, resolution limits, conformational averaging, crystal packing, etc.), force field imbalances, and real flexibility of the studied systems. These factors, and thus the simulation behavior, differ from system to system. The structural stability of simulated systems does not correlate with the size of buried interaction surface or experimentally determined binding affinities but reflects the type of protein-RNA recognition. Protein-RNA interfaces involving shape-specific recognition of RNA are more stable than those relying on sequence-specific RNA recognition. The differences between the protein force fields are considerably smaller than the uncertainties caused by sampling and starting structures. The ff12SB improves description of the tyrosine side-chain group, which eliminates some problems associated with tyrosine dynamics.

配列特異的RNA分解酵素の配列認識および分解制御機構

配列特異的RNA分解酵素の配列認識および分解制御機構

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins.

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.

Prevalent RNA recognition motif duplication in the human genome

The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.